Estradiol is one of a number of physiologically important steroid hormones among which minor differences in structure result in profoundly different biological activities [Cook, B. & Beastall, G. (1989). In "Steroid Hormones: a practical approach." Practical Approach Series (Rickwood, D. & Hames, B. D., eds.), Oxford University Press, Oxford. pp. 1-65.]. Measurement of estradiol levels in body fluids is important in clinical practice, for example to follow the menstrual cycle in women, and to monitor compliance and optimal dosing of estradiol administered as part of hormone replacement therapy in women at menopause. High levels of estradiol in men can be diagnostic of estrogen-secreting tumours such as Leydigs cell tumours, liver disease and hyperthyroidism.
Physiological concentrations of estradiol at particular stages in the menstrual cycle vary between individuals, but typically rise from around 0.18 nM at the lowest point to a peak of 1.5 nM prior to ovulation. These low levels of hormone make quantitative determination of estradiol difficult, placing a premium on the sensitivity and selectivity of assays. Antibody based detection methods are commonly used in diagnostic assays, but in the case of estradiol the antibody itself has proved to be the limiting factor.
Since steroids are not themselves immunogenic, they have to be coupled to a larger molecule in order to elicit an immune response in animals. However, it has been found that the antibodies raised in this way are often unable to recognise important structural differences in that part of the steroid attached to the carrier. The importance of specificity is not only in ensuring that the right hormone is measured, but also in discriminating between the biologically active form and metabolites inactivated by the liver [Cook, B. & Beastall, G. (1989)]. The inability to discriminate between closely related structures is manifested by the antibody having similar affinity for different steroids substituted at particular positions, and is often referred to as a `blind-spot` [Aravelo, J. H., Stura, E. A., Taussig, M. J. & Wilson, I. A. (1993). J. Mol. Biol. 231, 103-118].
Useful polyclonal antisera have nevertheless been obtained [Cook, B. & Beastall, G. (1989)], though affinity and cross-reactivity of sera is highly variable between animals, creating problems in standardizing assays, and many animals need be immunised before an antiserum with the required properties is obtained, limiting the supply of serum. All currently available diagnostic kits use polyclonal, antisera (e.g. Abbott, Amersham etc.) Consequently, many attempts have been made to generate monoclonal antibodies. Although some have had high affinities (typically 50 nM or better), none has had the requisite specificity. The well-characterised mouse monoclonal antibody DB3 [Aravelo, J. H. et al.] binds to progesterone with a dissociation constant of approximately 1 nM. This antibody provides an excellent example of the properties of an antibody raised against a steroid coupled to a protein; although DB3 is specific to the portion of the steroid furthest from the carrier, it shows very little specificity at the end of the steroid that was conjugated to the protein and will bind equally well when even large substituents are present at this site.
Phage display has previously been used to isolate a progesterone-binding antibody from a non immunised murine repertoire [Gram, H., Marconi, L., Barbas, C. F., Collet, T. A., Lerner, R. A. & Kang, A. S. (1992). Proc. Natl. Acad. Sci. (USA) 89, 3576-3580.]. The dissociation constant of the resulting antibody was only micromolar, and cross-reactivity to other steroids was not explored.